Periodontitis vaccine and related compositions and methods of use

ABSTRACT

An immunogenic composition, a periodontal vaccine formulation containing the immunogenic composition, and methods for treating or preventing periodontal disease are provided, where the methods involves administering an immunologically effective amount of the composition or vaccine formulation to a subject. The immunogenic composition contains at least one polypeptide that comprises: an Mfa1 antigen sequence that is substantially homologous to an immunogenic amino acid sequence from an Mfa1 fimbrilin protein of a  Porphyromonas  bacterium; and an HA1 antigen sequence, an HA2 antigen sequence, or both an HA1 antigen sequence and an HA2 antigen sequence, wherein the HA1 antigen sequence is substantially homologous to an immunogenic amino acid sequence from an RgpA Gingipain hemagglutinin domain 1 contained within an RgpA Gingipain protein of a  Porphyromonas  bacterium, and the HA2 antigen sequence is substantially homologous to an immunogenic amino acid sequence from an RgpA Gingipain hemagglutinin domain 2 contained within an RgpA Gingipain protein of a  Porphyromonas  bacterium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/035,242, filed Sep. 28, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/158,155, filed Oct. 11, 2018, now U.S. Pat. No.10,835,590, which issued Nov. 17, 2020, which claims priority under 35U.S.C. § 119(e)(1) to provisional U.S. Patent Application Ser. No.62/571,582, filed Oct. 12, 2017, the contents of each of which areincorporated herein by reference in its entirety.

SEQUENCE LISTING

The contents of the file named STRO_002_03US_SeqList_ST25.txt, which wascreated on May 4, 2021, and is 17.7 KB in size are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to the prevention and treatmentof periodontitis, and more particularly relates to a periodontal vaccinecomposition and a method for its use.

BACKGROUND

Periodontal diseases, collectively referred to as “periodontitis,” arecommonly occurring, yet complex chronic oral inflammatory diseases thatdestroy the soft and hard tissues supporting the teeth. If leftuntreated, the loss of the alveolar bone around the teeth can result inthe loosening and subsequent loss of teeth. Periodontal disease is amongthe most common human diseases of bacterial origin, with recent studiesindicating that approximately 60% of individuals over 40 years of age inthe United States have moderate or severe periodontitis and possessmeasurable oral bone loss. The prevalence of periodontitis increaseswith age; see Eke et al. (2012) J Dent. Res. 91(10): 914-920. Severegeneralized periodontal disease occurs in an estimated 5-20% ofindividuals globally (Burt et al. (2005) J. Periodontol. 76(8): 1406-19,often resulting in multiple tooth loss by middle age, and the economicburden of this disease is significant, with 2010 data estimating theeconomic impact at $54 billion in the US alone (Listl et al. (2015) J.Dent. Res. 94(10): 1355-61.

Periodontitis is characterized according to both severity and cause in aclassification system with seven recognized major categories: (1)gingival disease, or “gingivitis,” involving inflammation of thegingiva; (2) chronic periodontitis, a slowly progressive disease thatmay be either localized or generalized; (3) early onset, or “aggressive”periodontitis; (4) periodontitis associated with a systemic disease suchas diabetes mellitus, AIDS, and leukemia; (5) necrotizing periodontaldisease; (6) periodontal abscesses; and (7) periodontitis associatedwith endodontic lesions. The latter six categories are designated“destructive” periodontal diseases because the damage caused isirreversible. See Armitage (1999) Ann. Periodontol. 4(1): 1-6. Thesymptoms of periodontitis include inflamed or bleeding gums, gingivalrecession, pockets between the teeth and gums, and, in the case ofsevere periodontitis, loosening or loss of teeth. Treatments will dependon the extent and cause of the disease, and include scaling, rootplaning, antibiotic therapy, and surgery. Scaling and root planing oftenhave to be carried out multiple times, antibiotic therapy can beproblematic insofar as beneficial oral microbes can be killed along withthe pathogenic bacteria, and oral surgery is a generally undesirablesolution of last resort.

Periodontitis is initiated by the presence of keystone bacteria such asPorphyromonas gingivalis. Porphyromonas organisms possess an array ofmolecules contributing to its overall virulence, including fimbriae andgingipains (a group of cysteine proteases) that impact various aspectsof disease pathogenesis including attachment of bacteria to cells andother community microbes, development of inflammation, and microbialdysbiosis associated with periodontal disease; see Lamont et al. (1998)Microbiol. Mol. Biol. Rev. 62(4): 1244-63, and Bostanci et al. (2012)FEMS Microbiol. Lett. 333(1): 1-9. Several of these bacterial virulencefactors have been explored as potential targets for vaccine development.See, e.g., Lamont et al., supra; Arjunan et al. (2016) Mol. OralMicrobiol. 31(1): 78093; Takahashi et al. (2006) Cell Microbiol.8(5):738-57; Malek et al. (1994) J. Bacteriol. 176(4): 1-52-9); Gibsonet al. (2001) Infect. Immun. 69(12): 7959-63; and Evans et al. (1992)Infect. Immun. 60(7): 2926-35.

A vaccine to treat periodontitis—and possibly prevent periodontitis aswell—would eliminate the need for repeated clinical interventions and/ororal surgery. Development of an effective therapeutic and/orprophylactic vaccine for periodontal disease would be especially usefulas the disease occurs in a significant portion of the adult population.However, periodontitis is a multifactorial disease with factorsincluding bacterial composition of dental plaque, host genetic make-up,and environmental factors contributing unique barriers to a basicunderstanding of periodontal disease pathogenesis and the potential fortargeted vaccine development.

An ideal periodontitis vaccine would achieve therapeutic efficacy insubjects with periodontitis and be effective in the prophylactic contextas well. The need for aggressive clinical interventions would beeliminated, and the number of individuals suffering from periodontaldiseases would substantially decrease. In addition, an ideal vaccinewould be straightforward to manufacture using a cost-effective processamenable to large-scale production.

SUMMARY OF THE INVENTION

The invention is addressed to the aforementioned need in the art andprovides an immunogenic composition, a vaccine formulation comprisingthe composition, and methods for treating and preventing periodontaldisease.

In a first embodiment of the invention, an immunogenic composition isprovided comprising at least one polypeptide that comprises: (a) an Mfa1antigen sequence that is substantially homologous to an immunogenicamino acid sequence from an Mfa1 fimbrilin protein of a Porphyromonasbacterium; and (b) an HA1 antigen sequence, an HA2 antigen sequence, orboth an HA1 antigen sequence and an HA2 antigen sequence, wherein (i)the HA1 antigen sequence is substantially homologous to an immunogenicamino acid sequence from an RgpA Gingipain hemagglutinin domain 1 (alsoreferred to herein as “Gingipain HA1” or “HA1”) contained within an RgpAGingipain protein of a Porphyromonas bacterium, and (ii) the HA2 antigensequence is substantially homologous to an immunogenic amino acidsequence from an RgpA Gingipain hemagglutinin domain 2 (also referred toherein as “Gingipain HA1” or “HA1”) contained within an RgpA Gingipainprotein of a Porphyromonas bacterium.

In one aspect of this embodiment, the at least one polypeptide comprisesa first polypeptide that comprises: (a) an Mfa1 antigen sequence and anHA1 antigen sequence; (b) an Mfa1 antigen sequence and an HA2 antigensequence; or (c) an Mfa1 antigen sequence, an HA1 antigen sequence, andan HA2 antigen sequence. It will thus be appreciated that the firstpolypeptide may be a fusion protein that includes the Mfa1 antigensequence as well as the HA1 antigen sequence and/or the HA2 antigensequence. In a related aspect, the at least one polypeptide comprises(a) a first polypeptide comprising an Mfa1 antigen sequence; and (b) asecond polypeptide comprising an HA1 antigen sequence, an HA2 antigensequence, or both an HA1 antigen sequence and an HA2 antigen sequence.Thus, in this aspect the Mfa1 antigen sequence is within onepolypeptide, and the HA1 and HA2 antigen sequences are within a secondpolypeptide, which is a fusion polypeptide if both are present.

In another aspect of this embodiment, the at least one polypeptide ofthe immunogenic composition comprises a first polypeptide comprising anMfa1 antigen sequence, a second polypeptide comprising an HA1 antigensequence, and a third polypeptide comprising an HA2 antigen sequence.Thus, in this aspect, there are three distinct polypeptides eachcontaining one of the Mfa1, HA1 and HA2 antigen sequences.

In a related aspect, the Mfa1 antigen sequence, the HA1 antigensequence, and the HA2 antigen sequence are substantially homologous toan immunogenic amino acid sequence of the Mfa1 fimbrilin polypeptide,Gingipain HA1, and Gingipain HA2, respectively, of a Porphyromonasspecies selected from P. gingivalis, P. gulae, P. cangingivalis, P.gingivicanis, P. canoris, P. salivosa, and P. circumdentaria.

In another aspect of this embodiment, the Mfa1 antigen sequence, the HA1antigen sequence, and the HA2 antigen sequence are substantiallyhomologous to an immunogenic amino acid sequence of the Mfa1 fimbrilinpolypeptide, Gingipain HA1, and Gingipain HA2, respectively, of P.gingivalis.

In another aspect of this embodiment, the Mfa1 antigen sequence, the HA1antigen sequence, and the HA2 antigen sequence are substantiallyhomologous to an immunogenic amino acid sequence of the Mfa1 fimbrilinpolypeptide, Gingipain HA1, and Gingipain HA2, respectively, of P.gulae.

In another embodiment of the invention, a periodontitis vaccineformulation is provided that comprises an immunogenic composition asdescribed above and a pharmaceutically acceptable excipient. In theusual instance, the formulation contains at least one excipient, wherethe at least one excipient is selected from vehicles, solubilizers,emulsifiers, stabilizers, preservatives, isotonicity agents, buffersystems, dispersants, diluents, viscosity modifiers, and absorptionenhancers. The vaccine may, in addition or in the alternative, includeat least one adjuvant.

In one aspect of this embodiment, the vaccine formulation is formulatedas sterile injectable solution. In a related aspect of this embodiment,the vaccine formulation is formulated as a lyophilized composition to berehydrated prior to use.

In another embodiment, a method is provided for immunizing a subjectagainst periodontal disease by administering to the subject animmunologically effective amount of an immunogenic composition of theinvention. In one aspect of this embodiment, the method involvestreating periodontitis in a subject exhibiting symptoms ofperiodontitis. In another aspect of this embodiment, the method involvesreducing the risk of periodontitis developing in a subject, who may havea predisposition to developing periodontitis, including moderate tosevere periodontitis, where the predisposition is associated with a riskfactor such as age, genetic predisposition, an immunocompromised state,a systemic disease that increases the risk of developing periodontitis,the presence of endodontic lesions or abscesses, or other risk factors.Examples of systemic diseases that increase the risk of developingmoderate to severe periodontitis include diabetes mellitus, AIDS,leukemia, and Down's syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the SDS-PAGE analysis of purified polypeptides Mfa1,HA1, and HA2 generated by cell-free protein synthesis.

FIG. 2 provides the serum IgG EC₅₀ values against P. gingivalis Mfa1,HA1, and HA2. Groups of animals G1-G6 served as controls or experimentalgroups, and serum samples were collected from animals immediately priorto oral challenge (Post-Vax; open bars) or at sacrifice (filled bars),and molecule-specific IgG EC₅₀ values were calculated from ELISA dataagainst P. gingivalis (A) Mfa1, (B) HA1, and (C) HA2.

FIG. 3 shows the results of the experiments evaluating the effect of theperiodontitis vaccine formulation on oral bone loss in vivo.

DETAILED DESCRIPTION OF THE INVENTION

1. Terminology and Definitions:

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Specific terminology of particularimportance to the description of the present invention is defined below.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, “a polypeptide” refers not onlyto a single polypeptide but also to a combination of two or moredifferent polypeptides that may or may not be combined, “an adjuvant”refers to a single adjuvant as well as to two or more adjuvants that maybe separate or combined in a single composition, and the like.

A “biomolecule,” also referred to herein as a “biological molecule,” isany organic molecule, whether naturally occurring, recombinantlyproduced, chemically synthesized in whole or in part, or chemically orbiologically modified, that is, was or can be a part of a livingorganism. The term encompasses, for example, polypeptides, peptidefragments, amino acids, polysaccharides, lipids, and the like.

The term “polypeptide” is intended to include any structure comprised ofone or more amino acids, and thus includes dipeptides, oligopeptides,polypeptides, polypeptide fragments, and proteins. The amino acidsforming all or a part of a polypeptide may be any of the twentyconventional, naturally occurring amino acids, i.e., alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine(Y), as well as non-conventional amino acids such as isomers andmodifications of the conventional amino acids, e.g., D-amino acids,non-protein amino acids, post-translationally modified amino acids,enzymatically modified amino acids, β-amino acids, constructs orstructures designed to mimic amino acids (e.g., α,α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, β-alanine, naphthylalanine,3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, andnor-leucine), and other non-conventional amino acids, as described, forexample, in U.S. Pat. No. 5,679,782 to Rosenberg et al. The polypeptidesdescribed herein may include one or more non-natural amino acids bearinga functional group that enables conjugation to a secondary antigen,e.g., a polysaccharide. Polypeptides can be (a) naturally occurring, (b)produced by chemical synthesis, (c) produced by recombinant DNAtechnology, (d) produced by biochemical or enzymatic fragmentation oflarger molecules, (e) produced by methods resulting from a combinationof methods (a) through (d) listed above, or (f) produced by any othermeans for producing peptides, such as cell-free protein synthesis,described infra.

The terms “sequence identity,” “percent sequence homology,” and“sequence homology,” in the context of a polymeric biomolecule sequence,e.g., a polypeptide sequence, refer to two or more sequences that arethe same or have a specified percentage of amino acid residues (ornucleotides, or other types of monomer units making up the polymericbiomolecule) that are the same, when compared and aligned for maximumcorrespondence over a given length (comparison window), as measuredusing a sequence comparison algorithm, e.g., BLASTP or theSmith-Waterman homology search algorithm. In the present context, thepercent sequence homology may be determined over the full-length of thebiomolecule or just a portion. One method for calculating percentsequence homology is the BLASTP program having its defaults set at awordlength (W) of 3, an expectation (E) of 1 0, and the BLOSUM62 scoringmatrix; see, e.g., Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA89:10915. Exemplary determination of sequence alignment and % sequenceidentity employs the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using the default parametersprovided. If these preferred methods of calculating sequence identitygive differing amounts, the method giving the higher sequence identitycontrols.

The term “substantially homologous” refers to a percent sequencehomology over a given length (e.g., “x” amino acids of a polypeptide) ofat least about 50%, thus including, for example, at least about 75%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 99%, and 100%.

“Recombinant” polypeptides refer to polypeptides produced by recombinantDNA techniques, i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. “Synthetic” polypeptidesare those prepared by chemical synthesis.

As used herein, the term “immunogenic ” refers to the ability of anantigen (e.g., a polypeptide), to elicit an immune response, either ahumoral or cellular immune response, and preferably both. In a preferredembodiment, the subject will display either a therapeutic or protectiveimmunological response to administration of an “effective amount” or“immunologically effective amount” of an immunogenic composition hereinsuch that resistance to new infection will be enhanced and/or theclinical severity of the periodontal disease will be reduced. Theimmunological response will normally be demonstrated by alleviation orelimination of at least one symptom associated with the infection.

As used herein, when the term “purified” is used in reference to amolecule, it means that the concentration of the molecule being purifiedhas been increased relative to the concentration of the molecule in itsnatural environment. The term may also refer to purification of achemically synthesized molecule from a reaction mixture in which themolecule has been generated as a reaction product. As used herein, whenthe term “isolated” is used in reference to a molecule, the term meansthat the molecule has been removed from its native environment. Forexample, a polynucleotide or a polypeptide naturally present in a livingorganism is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials in its natural state is“isolated.” An isolated moiety, whether separated from a nativeenvironment or from a non-natural environment (e.g., recombinantexpression, cell-free expression, chemical synthesis, etc.), ispreferably are at least about 1% pure, 5% pure, 10% pure, 20% pure, 30%pure, 40% pure, 50% pure, 60% pure, 70% pure, 80% pure, 90% pure, 95%pure, or 99% pure, or they may be 100% pure. As used herein, the term “%pure” indicates the percentage of a composition that is made up of themolecule of interest, by weight.

As used herein, the term “molecular weight” of a polypeptide or otherbiomolecule refers to molecular weight calculated by size exclusionchromatography (SEC) combined with multiangle laser light scattering(MALS).

The term “treating” refers to therapeutic treatment by theadministration of an immunogenic composition or vaccine formulation ofthe invention, where the object is to lessen or eliminate infection. Forexample, “treating” may include directly affecting, suppressing,inhibiting, and eliminating infection, as well as reducing the severityof, delaying the onset of, and/or reducing symptoms associated with aninfection. Unless otherwise indicated explicitly or implied by context,the term “treating” encompasses “preventing” (or prophylaxis orprophylactic treatment) where “preventing” may refer to reducing therisk that a subject will develop an infection, delaying the onset ofsymptoms, preventing relapse of an infection, or preventing thedevelopment of infection.

2. Immunogenic Composition and Vaccine Formulation:

In a first embodiment of the invention, an immunogenic composition isprovided that includes at least one polypeptide that comprises: (a) anMfa1 antigen sequence that is substantially homologous to an immunogenicamino acid sequence from an Mfa1 fimbrilin protein of a Porphyromonasbacterium; and (b) an HA1 antigen sequence and/or an HA2 antigensequence, wherein the HA1 antigen sequence is substantially homologousto an immunogenic amino acid sequence from a Porphyromonas GingipainHA1, and the HA2 antigen sequence is substantially homologous to animmunogenic amino acid sequence from a Porphyromonas Gingipain HA2. Theat least one polypeptide may be a fusion protein that contains the Mfa1antigen sequence, the HA1 antigen sequence, and the HA2 antigensequence. The at least one polypeptide may also be a fusion protein thatcontains the Mfa1 antigen sequence and the HA1 antigen sequence, or afusion protein that contains the Mfa1 antigen sequence and the HA2antigen sequence. In a variation on such an embodiment, the at least onepolypeptide may comprise a first polypeptide that includes the Mfa1antigen sequence, and a second polypeptide that includes either or boththe HA1 antigen sequence and the HA2 antigen sequence.

In a preferred embodiment, however, the at least one polypeptide in theimmunogenic composition comprises two distinct polypeptides: a firstpolypeptide containing the Mfa1 antigen sequence and a secondpolypeptide containing the HA1 antigen sequence or the HA2 antigensequence. In another preferred embodiment, the at least one polypeptidein the immunogenic composition comprises three distinct polypeptides: afirst polypeptide containing the Mfa1 antigen sequence, a secondpolypeptide containing the HA1 antigen sequence, and a third polypeptidecontaining the HA2 antigen sequence.

As discussed above, the Mfa1 antigen sequence is substantiallyhomologous to an immunogenic amino acid sequence from an Mfa1 fimbrilinprotein of a Porphyromonas bacterium, the HA1 antigen sequence issubstantially homologous to an immunogenic amino acid sequence from aPorphyromonas Gingipain HA1, and the HA2 antigen sequence issubstantially homologous to an immunogenic amino acid sequence from aPorphyromonas Gingipain HA2. The immunogenic sequences within thesethree antigens can collectively or individually be the full the fulllength protein or domain (i.e., Mfa1 protein, RgpA Gingipainhemagglutinin domain 1, and/or RgpA Gingipain hemagglutinin domain 2),or a portion (or fragment) of such protein or domain so long as theportion selected results in compositions that possess the ability togenerate a therapeutic or prophylactic immunogenic response to aPorphyromonas bacterium infection. Usually these immunogenic portions orfragments of the full protein or domain are at least 20 amino acidresidues in length. Provided the desired immunogenic properties aremaintained, the length of the protein or domain sequence upon which theantigen sequence is based is a matter of design choice and can be atleast 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acidresidues, up to and including the full-length protein or domain. Theantigenic sequences comprised within the polypeptides of the presentinvention are, therefore, substantially homologous to these immunogenicamino acid sequences from the full-length or portion of the nativeprotein or domain sequences (i.e., Mfa1 fimbrilin protein, RgpAGingipain hemagglutinin domain 1, and/or RgpA Gingipain hemagglutinindomain 2). Typically, usually for reasons related to the methodology orefficiency of polypeptide production, the antigenic sequences comprisedwithin the polypeptides of the present invention are not exact copies ofthe native immunogenic sequence to which they correspond. For example,an N-terminal methionyl, which may be treated as outside the antigenicsequence to calculate maximum percent identity or homology, is oftenpresent due to the addition of a start codon. Additions, deletions andsubstitutions (often conservative substitutions) can also occur provideduseful immunogenic properties are still present in the polypeptide. Itcan be appreciated, therefore, that the present invention may be carriedout with polypeptides comprising (either within it or in its entirety)an amino acid residue sequence representing an antigenic sequence (i.e.,the Mfa1 antigen sequence, HA1 antigen sequence, or HA2 antigensequence) that is substantially homologous to an immunogenic amino acidresidue sequence found within the corresponding protein or domain,wherein the immunogenic amino acid residue sequence is either thefull-length protein or domain, or a portion or fragment thereof. Theseimmunogenic amino acid residue sequences against which the substantialhomology of the antigenic sequences are measured are individually eitherthe full-length protein or domain, or a portion thereof that is at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 60, at least 70, at least 80, at least 90, or 100amino acid residues in length. Typically, these antigenic sequences arehomologous to the immunogenic amino acid residue sequence against whichthe substantial homology of the antigenic sequence is measured at alevel of at least 50%, at least 75%, at least 80%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100%. Routine testing inanimals or humans can demonstrate readily whether compositions of thepresent invention based on portions of the full-length bacterialproteins or domains generate a therapeutic or prophylactic immunogenicresponse to infection by the bacteria in question.

Mfa1 fimbrilin protein is from a Porphyromonas bacterium, and the RgpAGingipain hemagglutinin domains 1 and 2 are contained within an RgpAGingipain protein that is also from a Porphyromonas bacterium, where thePorphyromonas bacterium may be any of various Porphyromonas species,including P. gingivalis, P. gulae, P. cangingivalis, P. gingivicanis, P.canoris, P. salivosa, and P. circumdentaria.

In a preferred embodiment, the Mfa1 fimbrilin protein is from P.gingivalis. Administration of an immunologically effective amount of acomposition containing at least one polypeptide with an Mfa1 antigensequence in this context will induce an immune response in whichanti-Mfa1 antibodies are generated, disrupting one or more of thepathogenic pathways by which a P. gingivalis infection proceeds. In thisembodiment, the Mfa1 antigen sequence is substantially homologous to animmunogenic amino acid sequence from an Mfa1 fimbrilin protein of a P.gingivalis bacterium, such as from the P. gingivalis Mfa1 fimbrilinprotein having the amino acid of SEQ ID NO:1.

The P. gingivalis Mfa1 fimbrilin polypeptide, prepared as described inthe Experimental Section, infra, contains 552 amino acids (this andfollowing SEQ IDs include the addition of an N-terminal methionyl from astart codon used in the cell-free synthesis described below) and has amolecular weight of 60,018, with the amino acid sequence of SEQ ID NO: 1reproduced for convenience below:

MGNGPDPDNAAKSYMSMTLSMPMGSARDGQNQDNPQYNFVGEWAGKDKIEKVSIYMVPQGGPGLVESAEDLDEGTYYDAPTQEAGSNNVILKPKKGIKVNSAVGKTVKVYVVLNDIAGKAKALLANVNAVDFEAKFKEVIELSTQAQALGTVADGPNPATAAGKIAKKNGVDNETIMMTCFEPSAPLTIEAAVSEANAIAGVKNQAKVTVERSVARAMVSTKAESYEIKATTQIGSIAAGDVLATVSDIRWVVAQGERKQYLSKKRGTVPENTWVTPGSDYISTNANFHAQATMYYDYTGLWDDHNADPTMVSGTKVPTLANYQLQDVTDELAQRLSGKFLLPNTHKSGIDAATSHYKRGNTAYVLVRAKFTPKKEAFIDKGKDYTDGTPVPEYTDGDDFFVGENGQFYVSMKSVTDPKVGGVAGMKAHKYVKGKVLYYAWLNPSTTSPDSWWNSPVVRNNIYHIHIKSIKKLGFNWNPLVPNPQNPNDPNGPINPNNPDPNPDEPGTPIPTDPEQPLPDQDTFMSVEVTVLPWKVHSYEVDL

In this embodiment, in which the Mfa1 antigen sequence is substantiallyhomologous to an immunogenic amino acid sequence from an Mfa1 fimbrilinprotein from P. gingivalis, it is preferred, although not essential,that the HA1 antigen sequence be substantially homologous to animmunogenic amino acid sequence from an RgpA Gingipain hemagglutinindomain 1 (HA1) contained within a P. gingivalis RgpA Gingipain protein,where, for example, the immunogenic amino acid sequence may be from theHA1 having SEQ ID NO: 2. Analogously, it is preferred, in thisembodiment, that the HA2 antigen sequence be substantially homologous toan immunogenic amino acid sequence from an RgpA Gingipain hemagglutinindomain 2 (HA2) contained within a P. gingivalis RgpA Gingipain protein,where the immunogenic amino acid sequence may be from the HA2 having SEQID NO:3.

P. gingivalis Gingipain HA1, prepared as described infra, contains 176amino acids and has a molecular weight of 19,059, with the amino acidsequence of SEQ ID NO: 2 reproduced for convenience below:

MLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTFWVCAQDANYASEHYAVYASSTGNDASNFTNALLEETITAKGVRSPEAIRGRIQGTWRQKTVDLPAGTKYVAFRHFQSTDMFYIDLDEVEI

P. gingivalis Gingipain HA2, also prepared as described infra, contains442 amino acids and has a molecular weight of 48,299, with the aminoacid sequence of SEQ ID NO: 3, reproduced below:

MFTETFESSTHGEAPAEWTTIDADGDGQDWLCLSSGQLDWLTAHGGTNVVASFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKVCVNVTINPTQFNPVKNLKAQPDGGDVVLKWEAPSGKRGELLNEDFEGDAIPTGWTALDADGDGNNWDITLNEFTRGERHVLSPLRASNVAISYSSLLQGQEYLPLTPNNFLITPKVEGAKKITYKVGSPGLPQWSHDHYALCISKSGTAAADFEVIFEETMTYTQGGANLTREKDLPAGTKYVA FRHYNCTDVLGIMIDDVVI

Combining the Mfa1 antigen sequence with either or both the HA1 antigensequence and the HA2 antigen sequence in one or more polypeptides in asingle immunogenic composition is believed to target multiple mechanismsinvolved in the pathogenic progression of periodontal disease associatedwith Porphyromonas infection, i.e., mechanisms associated with the Mfa1fimbrilin protein as well as the RgpA gingipain proteins.

In another embodiment, the Mfa1 fimbrilin protein is from P. gulae. Inthis case, the Mfa1 antigen sequence is substantially homologous to animmunogenic amino acid sequence from an Mfa1 fimbrilin protein of a P.gulae bacterium, such as from the P. gulae Mfa1 fimbrilin protein havingthe amino acid of SEQ ID NO:4. As explained with respect to P.gingivalis, the HA1 antigen sequence and the HA2 antigen sequence shouldbe substantially homologous to immunogenic amino acid sequences from anRgpA hemagglutinin domain 1 and an RgpA hemagglutinin domain 2,respectively, contained within an RgpA Gingipain protein of a P. gulaeorganism. The immunogenic amino acid sequence from the P. gulae HA1 maybe from SEQ ID NO: 5, and the immunogenic amino acid sequence from theP. gulae HA2 may be from SEQ ID NO: 6.

P. gulae Mfa1 fimbrilin polypeptide  (SEQ ID NO: 4):MGNGPDPDNAAKSYMSMTLSMPLGSARAGDGQDQPNPDYNYVGEWAGKDKIEKVSIYMVPQGGPGLVESAEDLDFSTYYDAPTQDPGSNNVILKPKKGIKVNSAVGKTVKVYVVLNDIAGKAKALLANVNAADFDAKFKEVIELSTQAEAVSQANAFNGTAAGKIAKKNGATDETIMMTCLQPSDALTIEAAVSEANAIAGVKNQAKVTVERSVARAMLSTKADTFEILAANQIGEIAAGSVLATITDIRWVVAQGERRQYLSKKRGTIQENTWVTPGSDFVPTSSTFHTNATEYYDYAGWEDHNTDPTVISGTQVPTLADYQLQNVTDELAQSLSGKFLLPNTHKSGTDAATSHYKRGNTAYVLIRAKFTPKKEAFIDKGKTYTDGTQVPEYEADQDFFVGENGQFYVSMKSVTDPKVGGVTGMKAHKYVKGKVLYYAWLNPSTTSPDTWWNSPVVRNNIYHIHIKSIKKLGFNWNPLVPDPNPNDPVNPNNPDPNPDEPGTPVPTDDPEQPLPDQDTFMSVEVTVLPWKVHSYEVDL P. gulae Gingipain HA1 (SEQ ID NO: 5):MTESFDGGIPATWTLIDADGDGHGWKHGKAPGVAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALNLPNGGKLTFWVCAQDAAYASEHYAVYASSTGNAASNFTNALLEETLTAKGVRSPEAIRGRVQGTWYQKTVDLPAGTKYVAFRHFQSTDMFYIDIDEVEI P. gulae Gingipain HA2 (SEQ ID NO: 6):MNAKRSELLNENFEGDDIPAGWTALDADGDGNNWGVQLNQFTRGEREALAPLRASNVAISYSSLNQGGGYLPLTPNNFLITPKVEGAKKISYKVGSPGNQSWSHDHYALCISKTGTAASDFEIIFEETMVYSQGGANFTREKDLPDGTKYVAFRHYNCTDVLAIVIDDVVITG

The present immunogenic compositions thus include:

(1) an immunogenic composition comprising (a) a P. gingivalis Mfa1antigen sequence that is substantially homologous to an immunogenicamino acid sequence from P. gingivalis Mfa1 fimbrilin protein having SEQID NO: 1, and (b) a P. gingivalis HA1 antigen sequence that issubstantially homologous to an immunogenic amino acid sequence from P.gingivalis Gingipain HA1, SEQ ID NO: 2;

(2) an immunogenic composition comprising (a) a P. gingivalis Mfa1antigen sequence that is substantially homologous to an immunogenicamino acid sequence from P. gingivalis Mfa1 fimbrilin protein having SEQID NO: 1, and (b) a P. gingivalis HA2 antigen sequence that issubstantially homologous to an immunogenic amino acid sequence from P.gingivalis Gingipain HA2, SEQ ID NO: 3;

(3) an immunogenic composition comprising (a) a P. gingivalis Mfa1antigen sequence that is substantially homologous to an immunogenicamino acid sequence from P. gingivalis Mfa1 fimbrilin protein having SEQID NO: 1, (b) a P. gingivalis HA1 antigen sequence that is substantiallyhomologous to an immunogenic amino acid sequence from P. gingivalisGingipain HA1, SEQ ID NO: 2, and (c) a P. gingivalis HA2 antigensequence that is substantially homologous to an immunogenic amino acidsequence from P. gingivalis Gingipain HA2, SEQ ID NO: 3;

(4) an immunogenic composition comprising (a) a P. gulae Mfa1 antigensequence that is substantially homologous to an immunogenic amino acidsequence from P. gulae Mfa1 fimbrilin protein having SEQ ID NO: 4, and(b) a P. gulae HA1 antigen sequence that is substantially homologous toan immunogenic amino acid sequence from P. gulae Gingipain HA1, SEQ IDNO: 5;

(5) an immunogenic composition comprising (a) a P. gulae Mfa1 antigensequence that is substantially homologous to an immunogenic amino acidsequence from P. gulae Mfa1 fimbrilin protein having SEQ ID NO: 4, and(b) a P. gulae HA2 antigen sequence that is substantially homologous toan immunogenic amino acid sequence from P. gulae Gingipain HA1, SEQ IDNO: 6; and

(6) an immunogenic composition comprising (a) a P. gulae Mfa1 antigensequence that is substantially homologous to an immunogenic amino acidsequence from P. gulae Mfa1 fimbrilin protein having SEQ ID NO: 4, (b) aP. gulae HA1 antigen sequence that is substantially homologous to animmunogenic amino acid sequence from P. gulae Gingipain HA1, SEQ ID NO:5, and (c) a P. gulae HA2 antigen sequence that is substantiallyhomologous to an immunogenic amino acid sequence from P. gulae GingipainHA1, SEQ ID NO: 6.

In a preferred embodiment, the composition comprises an immunogeniccomposition suitable for incorporation into a vaccine formulation. Atleast one polypeptide containing a selected antigen sequence, asdescribed above, is preferably incorporated into the composition inisolated or purified form, where the terms “isolated” and “purified” aredefined earlier herein. The amount of the at least one polypeptide inthe immunogenic composition is a sufficient and effective amount togenerate a therapeutic or prophylactic immune response in a subject,i.e., an amount rendering the composition as a whole immunogenic. Thus,administration of an immunologically effective dose of the immunogeniccomposition to a subject, in a vaccine formulation, will elicit animmune response as explained in part (I) of this section, preferably aresponse that serves to inhibit the progression of, or prevent the onsetof, periodontal disease associated with a Porphyromonas infection. Therelative amounts of each polypeptide in the composition may vary a greatdeal. However, the composition is generally formulated with the selectedpolypeptides—for example, (a) a first polypeptide comprising the Mfa1antigen sequence and a second polypeptide comprising the HA1 antigensequence, the HA2 antigen sequence, or both; or (b) a first polypeptidecomprising the Mfa1 antigen sequence, a second polypeptide comprisingthe HA1 antigen sequence, and a third polypeptide comprising the HA2antigen sequence—combined in amounts corresponding to a weight ratio ofeach polypeptide in the composition to each other polypeptide in thecomposition in the range of about 1:5 to about 5:1, typically in therange of about 1:3 to about 3:1, for example in the range of about 1:1.5to about 1.5:1, including about 1:1. In a preferred embodiment, theweight ratio of each polypeptide in the composition to each otherpolypeptide in the composition is in the range of 1:5 to 5:1, typically1:3 to 3:1, such as 1:1.5 to 1.5:1, and including 1:1. Accordingly, theimmunogenic composition may be one of the following:

(1) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA1antigen sequence in a weight ratio ranging from 1:5 to 5:1;

(2) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA1antigen sequence in a weight ratio ranging from 1:3 to 3:1;

(3) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA1antigen sequence in a weight ratio ranging from 1:1.5 to 1.5:1;

(4) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA1antigen sequence in a weight ratio of about 1:1;

(5) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA2antigen sequence in a weight ratio ranging from 1:5 to 5:1;

(6) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA2antigen sequence in a weight ratio ranging from 1:3 to 3:1;

(7) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA2antigen sequence in a weight ratio ranging from 1:1.5 to 1.5:1;

(8) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence and a second polypeptide comprising the HA2antigen sequence in a weight ratio of about 1:1;

(9) a composition formulated by combining a first polypeptide comprisingthe Mfa1 antigen sequence, a second polypeptide comprising the HA1antigen sequence, and a third polypeptide comprising the HA2 antigensequence, wherein the weight ratio of each polypeptide in thecomposition to each other polypeptide in the composition is in the rangeof 1:5 to 5:1;

(10) a composition formulated by combining a first polypeptidecomprising the Mfa1 antigen sequence, a second polypeptide comprisingthe HA1 antigen sequence, and a third polypeptide comprising the HA2antigen sequence, wherein the weight ratio of each polypeptide in thecomposition to each other polypeptide in the composition is in the rangeof 1:3 to 3:1;

(11) a composition formulated by combining a first polypeptidecomprising the Mfa1 antigen sequence, a second polypeptide comprisingthe HA1 antigen sequence, and a third polypeptide comprising the HA2antigen sequence, wherein the weight ratio of each polypeptide in thecomposition to each other polypeptide in the composition is in the rangeof 1:1.5 to 1.5:1; and

(12) a composition formulated by combining a first polypeptidecomprising the Mfa1 antigen sequence, a second polypeptide comprisingthe HA1 antigen sequence, and a third polypeptide comprising the HA2antigen sequence, wherein the weight ratio of each polypeptide in thecomposition to each other polypeptide in the composition is about 1:1,such that the weight ratios of the three polypeptides are about 1:1:1.

Additional antigens: In addition to the at least one polypeptidecomprising the Mfa1 antigen sequence and the HA1 antigen sequence and/orthe HA2 antigen sequence, the immunogenic composition may contain one ormore additional antigens. An additional antigen may be one that inducesan antibody response that targets Porphyromonas pathogenic mechanismsand/or virulence factors, for example, the bacterial tyrosine kinasePtk1 (see Bainbridge (2010) Infect. Immun. 78(11): 4560-69) or thephosphoserine phosphatase enzyme SerB (see Wright et al. (2014)(MicrobiologyOpen 3(3): 383-94. Antigens may also be included in thecomposition that are directed toward pathogens other than Porphyromonasorganisms, such as organisms that tend to be present in themultimicrobial biofilm associated with the progression of periodontaldisease.

The at least one polypeptide of the immunogenic composition can beprepared in many ways, e.g., by solid phase or liquid phase chemicalsynthesis (in whole or in part), by digestion of longer polypeptidesusing proteases, by cell-based recombinant protein expression, bypurification from a cell culture (e.g. from recombinant expression),etc. A preferred method for preparing the polypeptides, however, is thescalable cell-free protein synthesis (“CFPS”) system, described in U.S.Pat. No. 9,040,253 to Roy et al., U.S. Pat. No. 9,650,621 to Thanos etal., and Murray et al. (2013) Current Opin. Chem. Biol. 17(3): 420-26,all of which are incorporated by reference herein. Cell-free synthesisof the Mfa1, HA1, and HA2 polypeptide antigens is described in detail inthe Examples below.

The invention also provides a vaccine formulation that comprises theimmunogenic composition in a sterile formulation for administration to asubject, e.g., as a suspension, solution or in lyophilized form to berehydrated prior to use. The vaccine formulation includes the at leastone polypeptide comprising the Mfa1 antigen sequence and the HA1 antigensequence and/or the HA2 antigen sequence; optional additional antigensas explained above; and at least one additional component selected fromadjuvants and excipients, as follows:

Adjuvants: The vaccine formulation may contain one or more adjuvants topotentiate the immune response to one or more antigens in theimmunogenic composition. Suitable vaccine adjuvants for incorporationinto the present formulation are described in the pertinent texts andliterature and will be apparent to those of ordinary skill in the art.The major adjuvant groups are as follows:

Mineral salt adjuvants, including alum-based adjuvants such as aluminumphosphate, aluminum hydroxide, and aluminum sulfate, as well as othermineral salt adjuvants such as the phosphate, hydroxide, and sulfatesalts of calcium, iron, and zirconium;

Saponin formulations, including the Quillaia saponin Quil A and the QuilA-derived saponin QS-21, as well as immune stimulating complexes(ISCOMs) formed upon admixture of cholesterol, phospholipid, and asaponin;

Bacteria-derived and bacteria-related adjuvants, including, withoutlimitation, cell wall peptidoglycans and lipopolysaccharides derivedfrom Gram negative bacteria such as Mycobacterium spp., Corynebacteriumparvum, C. granulosum, Bordetella pertussis, and Neisseria meningitis,such as Lipid A, monophosphoryl Lipid A (MPLA), other Lipid Aderivatives and mimetics (e.g., RC529), enterobacteriallipopolysaccharide (“LPS”), TLR4 ligands, and trehalose dimycolate(“TDM”);

Muramyl peptides such as N-acetyl muramyl-L-alanyl-D-isoglutamine(“MDP”) and MDP analogs and derivatives, e.g., threonyl-MDP and nor-MDP;

Oil-based adjuvants, including oil-in-water (01W) and water-in-oil (W/O)emulsions, such as squalene-water emulsions (e.g., MF59, AS03, AF03),complete Freund's adjuvant (“CFA”) and incomplete Freund's adjuvant(“IFA”);

Liposome adjuvants;

Microsphere adjuvants formed from biodegradable and non-toxic polymerssuch as a poly(α-hydroxy acid), a poly(hydroxy butyric) acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.;

Human immunomodulators, including cytokines, such as interleukins (e.g.IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons (e.g.interferon-y), macrophage colony stimulating factor, and tumor necrosisfactor;

Bioadhesives and mucoadhesives, such as chitosan and derivatives thereofand esterified hyaluronic acid and microspheres or mucoadhesives, suchas cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol,polyvinyl pyrrolidone, polysaccharides and carboxymethylcellulose;

Imidazoquinolone compounds, including Imiquamod and homologues thereof,e.g., Resiquimod;

TLR-9 agonists, such as Hsp90 and oligodeoxynucleotides containingunmethylated CpG motifs (see, e.g., Bode et al. (2011) Expert Rev.Vaccines 10(4): 499-511); and

Carbohydrate adjuvants, including the inulin-derived adjuvants gammainulin and algammulin, and other carbohydrate adjuvants such aspolysaccharides based on glucose and mannose, including glucans,dextrans, lentinans, glucomannans, galactomannans, levans, and xylans.

Exemplary adjuvants herein include alum-based salts such as aluminumphosphate and aluminum hydroxide.

The vaccine formulation also includes at least one excipient, andusually two or more excipients, to serve any of a number of functions,where the excipients are immunologically and pharmacologically inertcomponents that are “pharmaceutically acceptable.” A “pharmaceuticallyacceptable” component herein is one that (1) can be included in avaccine formulation administered to a subject without causingsignificant unwanted biological effects or interacting in a deleteriousmanner with any of the other components of the formulation; and (2)meets the criteria set out in the Inactive Ingredient prepared by theU.S. Food and Drug Administration, and, preferably, has also beendesignated “Generally Regarded as Safe” (“GRAS”). The type of excipientor excipients incorporated into a vaccine formulation herein willdepend, in part, on the selected mode of administration and theparticular formulation type or dosage form, e.g., injectable liquidformulations, intranasal spray formulations, or the like; modes ofadministration and corresponding formulations are discussed infra. Ingeneral, however, inert components that can be advantageouslyincorporated into the vaccine formulation of the invention include,without limitation, vehicles, solubilizers, emulsifiers, stabilizers,preservatives, isotonicity agents, buffer systems, dispersants,diluents, viscosity modifiers, absorption enhancers, and combinationsthereof. A thorough discussion of pharmaceutically acceptable inertadditives is available in Gennaro (2000) Remington: The Science andPractice of Pharmacy. 20th ed., ISBN: 0683306472.

3. Administration and Use:

The immunogenic composition of the invention is useful in a method forimmunizing a subject against periodontal disease, where the methodinvolves administering to the subject an immunologically effectiveamount of a periodontal vaccine formulation comprising an immunogeniccomposition as described herein.

The subject may be a human or a non-human mammal, and the selection oftarget bacterium (which will be the source of the immunogenic amino acidsequences to which the antigen sequences correspond) may depend on thetype of subject. For example, an immunogenic composition according tothe present invention used to treat or prevent periodontitis in a humansubject will typically be targeted to P. gingivalis. As another example,an immunogenic composition according to the present invention used totreat or prevent periodontitis in a non-human mammal, where suchsubjects may be dogs, horses, dairy cattle, cats, or other mammals, willgenerally be targeted to P. gulae.

The method may involve administration of the immunogenic composition asa therapeutic vaccine, i.e., to treat a subject suffering fromperiodontitis. The method may also involve administration of theimmunogenic composition as a prophylactic vaccine, meaning that, forexample, the method reduces the risk of periodontitis (includingmoderate to severe periodontitis) developing in a subject and thus maypostpone or eliminate development of periodontitis. When the vaccine isused prophylactically, the subject may be predisposed to developingperiodontitis as a result of any number of risk factors, including age;a genetic predisposition; an immunocompromised state; a disease thatincreases the risk of developing moderate to severe periodontitis, suchas diabetes mellitus, AIDS, leukemia, Down's syndrome; or the presenceof endodontic lesions or abscesses. As an example, patients receivinganti-TNF therapy (i.e., taking a TNF inhibitor such as etanercept oradalimumab), such as in the treatment of rheumatoid arthritis orpsoriasis, often exhibit gingival inflammation and have an elevated riskof developing periodontitis.

The “immunologically effective amount” of the vaccine formulation is anamount that, either as a single dose or as part of a series of two ormore doses, is effective for treating or preventing periodontal disease,where “treating” and “preventing” are defined in part (1) of thissection. The amount administered will vary according to several factors,including the overall health and physical condition of the subject, thesubject's age, the capacity of the subject's immune system to synthesizerelevant antibodies, the form of the composition (e.g., injectableliquid, nasal spray, etc.), the taxonomic group of the subject (e.g.,human, non-human primate, non-primates, etc.), and other factors knownto the medical practitioner overseeing administration.

Administration of the immunogenic composition as a vaccine formulationcan be carried out using any effective mode of systemic delivery. Thecomposition is usually administered parenterally, such as by injection,including intravenous, intramuscular, intraperitoneal, interstitial, orsubcutaneous injection; injection may also be gingival, in which casethe vaccine formulation is injected directly into the gum. Thecomposition may, in addition, be administered transmucosally, such asvia the intranasal, sublingual, transbuccal, intravaginal, orintrarectal routes. Other modes of administration are also envisioned,however, and the invention is not limited in this regard. By way ofexample, other modes of administration include oral and transdermaldelivery as well as administration via inhalation or using a subdermalimplant.

The mode of administration largely dictates the type of formulation ordosage form that comprises the immunogenic composition. Compositionsformulated for parenteral administration include sterile aqueous andnonaqueous solutions, suspensions, and emulsions. Injectable aqueoussolutions contain the active agent in water-soluble form. Examples ofnonaqueous solvents or vehicles include fatty oils, such as olive oiland corn oil, synthetic fatty acid esters, such as ethyl oleate ortriglycerides, low molecular weight alcohols such as propylene glycol,synthetic hydrophilic polymers such as polyethylene glycol, liposomes,and the like. Parenteral formulations may also contain excipients suchas solubilizers, emulsifiers, stabilizers, preservatives, isotonicityagents, buffer systems, dispersants, diluents, viscosity modifiers,absorption enhancers, and combinations thereof Injectable formulationsare rendered sterile by incorporation of a sterilizing agent, filtrationthrough a bacteria-retaining filter, irradiation, or heat. They can alsobe manufactured using a sterile injectable medium. The immunogeniccomposition or individual components thereof may also be in dried, e.g.,lyophilized, form that may be rehydrated with a suitable vehicleimmediately prior to administration via injection.

Of the transmucosal routes, intranasal administration is generallyalthough not necessarily preferred. Intranasal formulations, includingintranasally administered vaccine formulations, are known in the art,and should be formulated with reference to the FDA's Guidance forIndustry: Nasal Spray and Inhalation Solution, Suspension, and SprayDrug Products. Intranasal formulations are liquids, i.e., solutions,emulsions, suspensions, or the like, for administration as sprays,intranasal injections, or drops, and can contain adjuvants andpharmaceutically acceptable excipients as above. Because of therelatively large size of the antigens in the formulation, systemicdelivery via the intranasal route requires incorporation of atransmucosal absorption enhancer in the immunogenic composition.Examples of suitable transmucosal absorption enhancers include, withoutlimitation, alkylsaccharides, cyclodextrins, and chitosans; see Maggio(2014) J. Excip. Food Chem. 5(2): 100-12; and Merkus et al. (1999) Adv.Drug Deliv. Rev. 36: 41-57. The concentration of enhancer is selected toensure that an immunologically effective amount of the formulationpasses through the nasal membrane and into the systemic circulation atan efficient transport rate. Various anatomical and physiologicalconsiderations dictating the composition and nature of an intranasalvaccine formulation are discussed, for example, by Aurora (October 2002)Drug Development & Delivery 2(7), incorporated by reference herein.

Other modes of administration and corresponding formulations include,without limitation: sublingual administration with a rapidly dissolvingdosage form such as a rapidly dissolving tablet; transbuccaladministration using a buccal patch or other buccal delivery system;intravaginal administration using a pessary, ointment, or cream;intrarectal delivery using a rectal suppository, ointment, or cream;transdermal administration using a transdermal patch or formulation;subdermal administration with an injected implant or pellet; inhalationusing a dry powder pulmonary formulation; and oral administration usingan oral dosage form such as a tablet, capsule, or the like.

As alluded to earlier herein, the vaccine formulation is administered toa subject within the context of an appropriate dosage regimen. Thecomposition may be administered once, or two or more times spaced outover an extended time period. For example, an initial, “prime” dose maybe followed by at least one “boost” dose. The time interval between theprime and the subsequent boost dose, and between boost doses, is usuallyin the range of about 2 to about 24 weeks, more typically in the rangeof about 2 to 12 weeks, such as 2 to 8 weeks, 3-6 weeks, etc. Regardlessof the mode of administration, e.g., intramuscular injection, gingivalinjection, intranasal administration, or the like, the volume of asingle dose of the vaccine will generally be in the range of about 1 μLto about 500 μL, typically in the range of about 1 μL to about 250 μL,more typically in the range of about 2.5 μL to about 200 μL, andpreferably in the range of about 5 μL to about 150 μL. It will beappreciated that the concentration of total antigen in the immunogeniccomposition corresponds to an immunologically effective dose of thecomposition per unit volume, working from the aforementioned dose volumeguidelines.

For ease of use, the immunogenic composition of the invention can beincorporated into a packaged product, or “kit,” including instructionsfor self-administration or administration by a medical practitioner. Thekit includes a sealed container housing a dose of the vaccineformulation, typically a “unit dose” appropriate for a single dosageevent that is immunologically effective. The vaccine may be in liquidform and thus ready to administer as an injection or the like, or it maybe in another form that requires the user to perform a preparationprocess prior to administration, e.g., hydration of a lyophilizedformulation, activation of an inert component, or the like. The kit mayalso include two or more sealed containers with the prime dose in afirst container and a boost dose in one or more additional containers,or a periodontitis vaccine formulation in a first container and avaccine directed against another infection, which may or may not berelated to the Porphyromonas infection, in another container.

It is to be understood that while the invention has been described inconjunction with a number of specific embodiments, the foregoingdescription as well as the experimental section that follows areintended to illustrate and not limit the scope of the invention. In thisregard, no attempt is made to show structural details of the inventionin more detail than is necessary for the fundamental understanding ofthe invention, the description taken with the drawings and/or examplesmaking apparent to those skilled in the art how the invention may beembodied in practice. This disclosure includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theelements of the invention described herein are encompassed by thedisclosure unless otherwise indicated herein or clearly contradicted bycontext.

EXPERIMENTAL Generation of Cell Free Extract:

Cell free extracts containing additional DsbC chaperone were prepared aspreviously described by Groff et al. (2014) Mabs 7(1):231-242. Briefly,E. coli strain SBJY001(see Yin et al. (2012) Mabs 6(3):671-678) wastransformed with a pACYC plasmid carrying tandem copies of the dsbCgene. Cells were grown, harvested and homogenized as described by Zawadaet al. (2011) Biotechnol. Bioeng. 108(7):1570-1578. Subsequentclarification via centrifugation yielded the extract used for subsequentcell free expression reaction.

Generation of Recombinant P. gingivalis Mfa1, HA1, HA2, andpurification: DNA sequences encoding the HA1, HA2 and Mfa1 fimbrilinproteins associated with P. gingivalis were codon optimized, synthesized(DNA 2.0; Menlo Park, Calif.) and cloned into the previously describedpYD317 vector [30]. Cell-free reactions were performed with the XpressCFTM CFPS system essentially as previously described (Yin et al., supra;Zimmerman et al. (2014) Bioconjug. Chem. 25(2):351-361). For expressionof HA1, HA2, and Mfa1 fimbrilin, reactions were performed with IAMpre-treatment, at 25° C., with the addition of oxidized glutathione (2mM) to create an oxidizing environment for the disulfide bonds.Expression of HA1 and HA2 was performed without IAM treatment of thecell extract; addition of reduced glutathione (8 mM) maintained areducing expression environment. After 16 h of reaction time, theexpressed proteins were isolated from the cell-free reaction mixturesusing his-tag affinity purification on Ni Sepharose resin (GELifesciences, Pittsburg, Pa.) per the manufacturer's recommendations.Further purification of the HA1, HA2, as well as Mfa1 fimbrilin proteinwas achieved via cation exchange chromatography on SP ImpRes resin (GELifesciences). Briefly, the Ni Sepharose elution pools were exchangedinto sodium citrate (50 mM), NaCl (50 mM), pH 4.5, and applied to acolumn equilibrated in the same buffer. Polished protein wassubsequently eluted via gradient elution to Tris (50 mM), NaCl (1 M), pH7.5. Similarly, HA2 was further purified via anion exchangechromatography on Q ImpRes (GE Lifesciences). Column equilibration andloading was performed in Tris (50 mM), NaCl (50 mM), pH 7.5, withsubsequent gradient elution to Tris (50 mM), NaCl (1M), pH 7.5.

After polishing chromatography, all proteins were dialyzed intoDulbecco's PBS and analyzed via SDS-PAGE. and intact mass analysis viaQ-TOF (Agilent, Santa Clara, Calif.). In the case of HA1, intact massanalysis conclusively showed that the cysteines were oxidized and hadformed the expected disulfide bond.

Cultivation of P. gingivalis and Bacterial Purity Assessment:

Porphyromonas gingivalis strain A7436 was handled as describedpreviously (Huang et al. (2015) Mol. Oral. Microbiol. 30(6):438-450). Inbrief, freezer stocks were plated on anaerobic blood agar plates, P.gingivalis were collected after three days of anaerobic growth at 37°C., harvested organisms were placed into sterile brain-heart infusionbroths supplemented with L-cysteine (0.75 g/L), hemin (5 mg/L), andmenadione (1 mg/L). After 24h, bacteria in log-phase growth wereharvested by centrifugation and suspended to 1×1010 CFU/mL in 2%carboxymethylcellulose in pyrogen-free saline for oral challenge (100μL/ challenge). For immunizations, broth grown P. gingivalis wereadjusted to 1×109 CFU/mL in injection-grade saline, and heat-killed (60°C. for 30 min.) prior to injection, and bacterial kill was confirmed byplating. Gram-staining was performed on all P. gingivalis broth culturesto ensure purity.

Mice, Immunizations, and Oral Challenge:

Six-week old female BALB/c mice (Charles River Laboratories, Wilmington,Mass.), were randomly separated into six groups (n=8/group), were housedin specific pathogen free facilities, and received water and food adlibitum. All live animal use was performed in accordance with IACUCapprovals. Groups included G1) non-immunized/no oral challenge control,G2) non-immunized/P. gingivalis oral challenge, G3) heat-killed P.gingivalis immunization/P. gingivalis oral challenge, G4) Mfa1+HA1+HA2combined immunization in alum/P. gingivalis oral challenge, G5)Mfa1+HA1+HA2 combined immunization in MPL/P. gingivalis oral challenge,and G6) Mfa1+HA1+HA2 combined immunization in injection-grade saline/P.gingivalis oral challenge. Prior to initiation of immunizations,baseline serum samples were obtained from each animal, and thenrespective groups of mice were immunized by intramuscular injection ofkilled P. gingivalis, or Mfa1+HA1+HA2 (5 μg of each protein/injection)suspended in either alum (Imject, ThermoFisher Sci, Rockford, Ill.),monophosphoryl lipid A (MPL; Sigma-Aldrich, St. Louis, Mo.), orinjection-grade saline. Subsequent intramuscular booster immunizationswere delivered 2-, and 4-weeks after the initial immunization. Two-weeksafter completion of immunization, a serum sample was obtained fromanimals immediately prior to oral challenge with P. gingivalis. Oralchallenge of mice was accomplished as reported previously (Gonzalez etal. (2003) Infect. Immun. 71(4):2283-2287). In brief, animals received10-day oral sulphamethoxazole/trimethoprim (Hi-Tech Pharmical,Amityville, N.Y.) in drinking water, followed by removal of antibioticsand a three-day rest. A P. gingivalis slurry (1×1010 CFU/ml+2%carboxymethylcellulose in injection-grade saline) was gently applied tothe gums of challenged mice using a syringe fitted with a feeding needle3-times over a 1-week period. Control animals included those that weremock challenged with 2% carboxymethylcellulose alone. After a 42-dayrest following completion of oral challenge, animals were sacrificed,terminal bleeds were obtained, and the head of each mouse was processedfor oral bone loss measurements. A final serum sample was collected fromeach animal at sacrifice, and all serum samples collected were stored at−80° C.

Detection of Mfa1-, HA1-, and HA2-Specific IgG in Mouse Sera:

Antigens (0.5 μg/mL) were plated at 4° C. overnight on Maxisorp plates(NUNC, Rochester, N.Y.), washed three times with PBS containing Tween-20(0.05%), and blocked with PBS with 1% BSA for a minimum of 1 h. Serial2-fold diluted serum samples from vaccinated mice (100 μL/well) wereadded to individual wells and incubated for 2 h at room temperature.Plates were washed, incubated with appropriate isotype specific antibodyconjugated to horseradish peroxidase (1:6000 dilution; Southern Biotech,Birmingham, Ala.), visualized with the addition of 100 μL of TMBsubstrate (Pierce, Rockford, Ill.) for 20-30 min, and reaction stoppedby the addition of 50 μL H2SO4 (1.0 M). Absorbance in each well wasmeasured at 450 nm minus the absorbance at 570 nm to correct for plateabnormalities. The resulting data for each sample were plotted to obtaina curve of the reciprocal dilution versus the A450-A570 measurement. Theantibody titer was determined as the midpoint of the dilution curve asdefined by EC50 calculations using Prism statistical analysis software(GraphPad Software, La Jolla, Calif.). The mean of the EC50 for eachcohort was determined to be the final antibody titer.

Measurement of Oral Bone Loss:

Oral bone levels were determined by morphometric analyses, as donepreviously (Gibson et al. (2001) Infect. Immun. 69(12): 7959-7963).After sacrifice, soft tissue was removed around the maxillary molars,and following extensive cleaning, the skulls were stained with methyleneblue. Prior to initiation of bone measurements, samples were blinded bya researcher not aware of the groupings. Oral bone measurements at themaxillary molars were obtained using a digital camera affixed to astereomicroscope from the alveolar bone crest (ABC) to the cementumenamel junction (CEJ) at 14 landmark sites (Baker et al. (1994) Arch.Oral Biol. 39(12): 1035-1040). Image analysis was performed using ImageJ(Schneider et al. (2012) Nat. Methods 9(7): 671-675) and onscreen pixellengths were converted to millimeters, and data obtained from eachanimal in a group were combined to achieve a group level meanlength±SEM.

Statistical Analysis:

Data were analyzed with Prism statistical analysis software (GraphPad).Comparison between groups was performed as indicated using unpairedStudent T test, or ANOVA with post-test analysis, and P<0.05 wasconsidered significant.

Results:

SDS-PAGE: The purified proteins generated by CFPS were denatured andadded to wells (3 μg/well), separated on 4-12% Bis-Tris gradient gels,and stained with coomassie blue. Results of the analysis of the proteinsgenerated under reducing conditions are shown in FIG. 1. Lane 1:molecular mass markers. Lane 2: Mfa1. Lane 3: HA1. Lane 4: HA2.

To determine whether the proteins delivered by intramuscular injectionelicited protein-specific IgG antibody responses, and to determinewhether different adjuvants (alum vs. MPL) influenced the elicited IgGresponse, sera were collected from groups of mice at the completion ofthe immunization period, and at sacrifice were tested for levels ofMfa1-, HA1- and HA2-specific IgG by ELISA. Titration curves for eachserum sample were converted to EC₅₀ values. As anticipated, seracollected from the non-immunized group of mice prior to oral challengepossessed low levels of IgG to Mfa1, HA1, and HA2. Sera collected frommice immunized with killed P. gingivalis A7436 elicited a nominalincrease in IgG specific to purified Mfa1, and HA2, with HA2>Mfa1. Forthe groups of mice immunized IM with the combined proteins suspended inalum, MPL, or injection-grade saline revealed that all mice receivingthe vaccine combination responded with antigen-specific IgG responses.Post-immunization levels of IgG to Mfa1 was most robust in MPL adjuvant;MPL>alum or saline. For HA, alum best facilitated molecule-specific IgGwith alum>MPL>saline, while for HA2 alum and MPL facilitatedantigen-specific IgG responsivity to similar levels and were bothgreater than that observed with saline.

Comparisons of IgG levels at sacrifice, revealed that the group ofnon-immunized mice oral challenged with P. gingivalis A7436 generatedslight elevation in specific IgG to Mfa1 and HA2, but not against HA1.Immunization with heat-killed P. gingivalis A7436 revealed a similarlow-level increase in in comparison to levels of IgG measuredimmediately prior to oral challenge, independent of adjuvant or saline,with the exception of measured IgG against HA1 from mice immunized withthe protein combination in saline.

FIG. 2 provides the serum IgG EC₅₀ values against P. gingivalis Mfa1,HA1, and HA2. Groups of animals G1-G6 served as controls or experimentalgroups, and serum samples were collected from animals immediately priorto oral challenge (Post-Vax; open bars) or at sacrifice (filled bars),and molecule-specific IgG EC5o values were calculated from ELISA dataagainst P. gingivalis (A) Mfa1, (B) HA1, and (C) HA2.

To understand if the protein combination could effectively limit theextent of P. gingivalis elicited oral bone loss, immunized animals weresubjected to P. gingivalis oral challenge. Groups of mice that were notimmunized, or immunized with killed P. gingivalis served as controls. Incomparison to mock challenged mice (G1), animals orally challenged withP. gingivalis A7436 (G2) developed oral bone loss as evidenced by anincrease in mean distance from ABC to CEJ (p<0.001). As anticipated,immunization with the killed preparation of P. gingivalis A7436 (G3)provided measurable protection from homologous organism-elicited oralbone loss (p<0.01). Groups of mice that received the combination proteinvaccine generated from a heterologous strain of P. gingivalis suspendedin either alum (G4) or MPL (G5) were protected from P.gingivalis-elicited oral bone loss (p<0.01 for each vs. P. gingivalisoral challenge alone. No differences in the level of protection (ABC toCEJ measurements) was observed between adjuvants, indicating thatintramuscular delivery of the vaccine candidate provided similarprotective responses (p>0.05). It was also observed that the group ofanimals immunized with the combination protein vaccine suspended insaline solution (G6) were also protected from P. gingivalis oralchallenge similar to that observed when the proteins were deliveredintramuscular with adjuvant (p>0.05 vs. alum or MPL adjuvants), and thelevel of protection was similar regardless of adjuvant employed, to thatprovided by heat-killed whole organism vaccine group (p>0.05 for all).

FIG. 3 shows the results of the experiments evaluating oral bone loss invivo. (A) BALB/c mice were randomized into groups (G1-6) and immunizedanimals received 3 intramuscular injections of combined protein cocktailin respective adjuvant, or in injection-grade saline at 2-week intervals(primary and 2 boosts; red arrows). Immunization control group (G3)received heat-killed P. gingivalis (equivalent to 1×10⁷ CFU/injection).All animals were placed on 10-day sulphamethoxazole/trimethoprim(antibiotics) in drinking water, followed by removal of antibioticsthree days prior to mock oral challenge (G1), or P. gingivalis oralchallenge (3× over a 1-week period; G2-6). After completion of oralchallenge (0 wks.), animals were allowed rest for six weeks and thensacrificed. FIG. 3 provides the results, showing the average distancebetween cementum enamel junction (CEJ) and alveolar bone crest (ABC) inmm±SEM, #=p<0.001 vs. G1 (unchallenged), ***=p<0.01 vs. G2 (P.gingivalis oral challenge only) (using ANOVA with Dunns multiplecomparisons).

1. An immunogenic composition comprising at least one recombinantpolypeptide wherein the at least one polypeptide comprises: (a) an Mfa1antigen sequence, wherein the Mfa1 antigen sequence having at least 90%sequence homology to SEQ ID NO: 4; and (b) an HA1 antigen sequence, anHA2 antigen sequence, or both an HA1 antigen sequence and an HA2 antigensequence, wherein (i) the HA1 antigen sequence comprises a sequencehaving at least 80% sequence homology to SEQ ID NO: 5, and (ii) the HA2antigen sequence comprises a sequence having at least 80% sequencehomology to SEQ ID NO:
 6. 2. The immunogenic composition according toclaim 1 wherein the at least one recombinant polypeptide comprises afirst recombinant polypeptide comprising: (a) the Mfa1 antigen sequenceand the HA1 antigen sequence; (b) the Mfa1 antigen sequence and the HA2antigen sequence; or (c) the Mfa1 antigen sequence, the HA1 antigensequence, and the HA2 antigen sequence.
 3. The immunogenic compositionaccording to claim 1 wherein the at least one recombinant polypeptidecomprises: (a) a first recombinant polypeptide comprising the Mfa1antigen sequence; and (b) a second recombinant polypeptide comprisingthe HA1 antigen sequence, the HA2 antigen sequence, or both the HA1antigen sequence and the HA2 antigen sequence.
 4. The immunogeniccomposition according to claim 1 wherein the at least one recombinantpolypeptide comprises: (a) a first recombinant polypeptide comprisingthe Mfa1 antigen sequence; (b) a second recombinant polypeptidecomprising the HA1 antigen sequence; and (c) a third recombinantpolypeptide comprising the HA2 antigen sequence. 5-7. (canceled)
 8. Theimmunogenic composition of claim 1, wherein the Mfa1 antigen sequencecomprises a sequence having at least 90% sequence homology to SEQ IDNO:4, the HA1 antigen sequence comprises a sequence having at least 80%sequence homology to SEQ ID NO:5 and the HA2 antigen sequence comprisesa sequence having at least 90% sequence homology to SEQ ID NO:6.
 9. Theimmunogenic composition of claim 1, wherein the Mfa1 antigen sequencecomprises a sequence having at least 90% sequence homology to SEQ IDNO:4, the HA1 antigen sequence comprises a sequence having at least 90%sequence homology to SEQ ID NO:5, and the HA2 antigen sequence comprisesa sequence having at least 80% sequence homology to SEQ ID NO:6. 10-13.(canceled)
 14. A vaccine formulation, comprising the immunogeniccomposition of claim 1 and at least one excipient. 15-23. (canceled) 24.The vaccine formulation of claim 14, wherein the at least one excipientis selected from vehicles, solubilizers, emulsifiers, stabilizers,preservatives, isotonicity agents, buffer systems, dispersants,diluents, viscosity modifiers, and absorption enhancers.
 25. The vaccineformulation of claim 24, further including an adjuvant. 26-27.(canceled)
 28. A method for immunizing a subject against periodontaldisease, comprising administering to the subject an immunologicallyeffective amount of the immunogenic composition of claim
 1. 29. A methodfor immunizing a subject against periodontal disease, comprisingadministering to the subject an immunologically effective amount of thevaccine formulation of claim
 14. 30. The method of claim 29, wherein theperiodontal disease is associated with a Porphyromonas bacteriumselected from P. gingivalis, P. gulae, P. cangingivalis, P.gingivicanis, P. canoris, P. salivosa, and P. circumdentaria. 31-32.(canceled)
 33. The method of claim 30, wherein the Porphyromonasbacterium is P. gulae.
 34. The method of claim 33, wherein the subjectis a non-human mammal.
 35. The method of claim 29, wherein the vaccineformulation comprises a sterile injectable solution and is administeredto the subject by injection. 36-41. (canceled)
 42. The method of claim29, wherein the subject exhibits symptoms of periodontitis and thevaccine formulation is administered as a therapeutic vaccine.
 43. Amethod for reducing the risk of periodontitis developing in a subject,the method comprising administering to the subject an immunologicallyeffective amount of the immunogenic composition of claim
 1. 44. A methodfor reducing the risk of periodontitis developing in a subject, themethod comprising administering to the subject an immunologicallyeffective amount of the vaccine formulation of claim
 14. 45. The methodof claim 44, wherein the subject has at least one risk factor ofdeveloping periodontitis.
 46. The method of claim 45, wherein the atleast one risk factor is selected from age, genetic predisposition, asystemic disease, the presence of endodontic lesions or abscesses, or acombination thereof.
 47. A method for reducing bone loss caused byperiodontal disease, comprising administering to a subject in need ofsuch treatment an immunologically effective amount of the composition ofclaim
 1. 48. A method for preventing bone loss caused by periodontaldisease, comprising administering to a subject in need of such treatmentan immunologically effective amount of the vaccine formulation of claim14.
 49. A method for reducing inflammation caused by periodontaldisease, comprising administering to a subject in need of such treatmentan effective amount of the immunogenic composition of claim
 1. 50. Amethod for reducing inflammation caused by periodontal disease,comprising administering to a subject in need of such treatment aneffective amount of the vaccine formulation of claim
 14. 51. A vaccineformulation, comprising the immunogenic composition of claim 4 and atleast one excipient.
 52. A method for immunizing a subject againstperiodontal disease, comprising administering to the subject animmunologically effective amount of the immunogenic composition of claim4.
 53. A method for immunizing a subject against periodontal disease,comprising administering to the subject an immunologically effectiveamount of the vaccine formulation of claim
 51. 54. The method of claim30, wherein the Porphyromonas bacterium is P. cangingivalis or P.gingivicani.
 55. The method of claim 51, wherein the subject is anon-human mammal.